Real-Time Quantitative Reverse Transcription PCR for Diagnosing Viral Infections

Abstract

Source: Marston, D. A. et al. Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis. J. Vis. Exp. (2019).

This video demonstrates a quantitative method for detecting viral infection using real-time reverse transcription PCR. The reverse transcription step converts the RNA to cDNA, which is then amplified via PCR cycles. The presence of viral RNA in the sample is confirmed by analyzing the amplification and dissociation curves of DNA obtained from the PCR.

Protocol

1. Quantification of RNA Using a Micro-volume Spectrophotometer

1. Ensure the settings of the spectrophotometer are set to RNA.
2. Use 1-2 µL of molecular-grade water to initialize the machine and set a baseline.
3. Use 1-2 µL of each test RNA sample to assess the RNA quantity.
4. Save the readings and document.
5. Adjust the RNA to 1 µg/µL, if required.
   NOTE: RNA must be kept on ice (or in a cool block) at all times. If RNA is obtained using a column, or bead-based method the RNA is usually less than 1 µg/µL. In this situation use the RNA neat.

Results

Table 1: Pan-lyssavirus real-time RT-PCR master mix reagents.

Reagent	μL/Reaction
Molecular-grade water	7.55
2x Universal RT PCR reaction mix	10
Primer Forward [20 μM]	0.6
Primer Reverse [20 μM]	.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Art Barrier pipette tips (various sizes)	Thermofisher	various
Centrifuge	Beckman	Allegra 21R	Rotor capable of holding 96 well plates required. Step 3.8.
Centrifuge (micro)	Sigma
Finnpipettes (to dispense 0.5-1000 µL)	Thermofisher	various
iTaq Universal SYBR Green One-Step RT-PCR kit	Bio-Rad	172-5150	Equivalent kits can be used if validated
MX3000P or MX3005P real-time PCR system	Stratagene	N/A	Equivalent machines can be used if validated
MicroAmp reaction plate base	Any suitable		Used to hold tube strip and plates securely.
Optically clear flat clear strips (8)	ABgene	AB-0866
Perfect fit frame (if using tube strips)	Stratagene	N/A	Specific to machine
Primers: for primer details see Table 2.			Ordered at 0.05 µmole scale HPLC purified.
Thermo-Fast 96-well plates, non-skirted	ABgene	AB-600
Thermo-Fast strips (8) Thermo-tubes	ABgene	AB-0452
Vortex machine / Whirlimixer	Fisons Scientific equipment	SGP-202-010J
Unless stated, alternative equipment can be used